Sterilization system and device

ABSTRACT

A system, device and method for sterilizing or decontaminating an object that by exposing the object to a sterilant gas comprised of one or more oxides of nitrogen, such as NO, NO 2 , NO 3 , N 2 O 3 , N 2 O 4 , N 2 O 5 , N 2 O and mixtures thereof. The source of the sterilant gas can be generated from a sterilant gas-generating composition or provided by a source of the sterilant gas.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. application Ser.No. 13/194,351, filed Jul. 29, 2011, which is a continuation of U.S.Ser. No. 11/477,513, filed Jun. 30, 2006, now U.S. Pat. No. 8,017,074,which is a continuation-in-part of PCT/US2005/000173, filed on Jan. 6,2005, which in turn claims the benefit and priority to U.S. provisionalapplication No. 60/534,395, filed on Jan. 7, 2004, U.S. provisionalapplication No. 60/575,421, filed Jun. 1, 2004, and U.S. provisionalapplication No. 60/564,589, filed Jul. 23, 2004, the entire contents ofall applications are incorporated herein by reference in theirentireties. The present application is related to U.S. ProvisionalApplication No. 60/542,298, filed Feb. 9, 2004, which is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to sterilization devices and methodologies usingnitric oxide and/or other oxides of nitrogen as the sterilant.Specifically, the invention relates to a device and method that usesnitric oxide and/or additional oxides of nitrogen for sterilizationpurposes.

BACKGROUND OF THE INVENTION

Steam autoclaving is the hospital standard for sterilizing most medicalinstruments. This method exposes materials to steam at 121° C. at 15-20lbs per square inch of pressure for 15-30 minutes. Killing is mediatedby heat denaturation of proteins, DNA, and subsequent interruption ofmetabolic functions. The method requires cumbersome equipment, a powersupply and plumbing, although benchtop models have Tillable watertables. Aside from these logistical problems, autoclaving is notsuitable for many plastics and other heat labile materials.

Sterilant gases can kill or control the growth of microbialcontaminations. Some of these sterilant gases include chlorine dioxide,sulfur dioxide, hydrogen peroxide, nitric oxide, nitrogen dioxide,carbon dioxide, hydrogen sulfide, ozone and ethylene oxide. One problemwith many of the sterilant gases is that they are explosive in highconcentrations (e.g. ethylene oxide, hydrogen peroxide, chlorinedioxide). Thus, storing, containing and using these gases in highconcentrations represent a hazard to the user. For safety reasons, thislimits the usable concentration of gas and creates an additionaldisadvantage. The concentration of the sterilant gas must be decreaseddue to safety concerns, while the exposure time must be increased toachieve effective sterilization.

Certain sterilant gases, such as chlorine dioxide, ozone and hydrogenperoxide are difficult and expensive to transport. Many of thesesterilant gases are powerful oxidizers. Oxidizing gases are expensiveand paperwork intensive to ship in bulk tanks, further complicatingtheir use. Gases, such as ozone and chlorine dioxide, must be generatedat or near the point of use. On-site plants for generating one suchsterilant gas, chlorine dioxide, are costly and require significantspace to implement.

Hamilton U.S. Pat. No. 6,607,696 describes a device for deliveringchlorine dioxide to disinfect or sterilize a liquid or an item containedin the liquid. The device uses a permeable sachet containing gasgenerating reactants, such as sodium chlorite and citric acid, where thesachet is a receptacle permeable to liquid and gas. Liquid can diffuseinto the receptacle to reach the gas generating reactants that thengenerate a gas, such as chlorine dioxide. The gas that diffuses out ofthe permeable sachet is not sealed from the environment/atmosphere.Multi-compartmental devices that employ gas-generating ingredientscontained in closed compartments that are permeable and permit thediffusion of liquids and gases through the compartments to producechlorine dioxide, such as the sachet and envelope compartments used inU.S. Pat. Nos. 6,602,466 and 6,607,696. Not only are these systemsexpensive and difficult to manufacture, but they do not contain thegenerated gases in a manner that prevents their unintended escape to theenvironment/atmosphere nor do they allow the user to predictably andcontrollably release the gas into a sealable container that is sealedwhen the contents are sterilized.

Thus, there is a need for methods and devices that generate sterilantgases at the point of use in a safe and efficient manner. There is afurther need for processes capable of producing significantconcentrations of sterilant gas without the danger of explosion oroxidative fire. There is a need to produce greater concentrations of NOin a short time period to allow a shorter exposure and make thesterilization process more efficient. There is also a need for a systemand method to generate small amounts of sterilant gas in an economicalmanner. The ability to economically generate small amounts of sterilantgases allows for easy transportation of the sterilizing system,imparting portability to the system not commonly found with traditionalsterilization devices and methods.

Given the problems with traditional gaseous sterilants anddisinfectants, there is a need for a sterilant gas generating system andmethod where the risk of explosion and oxidative fire is minimized, thatproduces the sterilant gas rapidly, safely, economically, and in ascaleable manner. There is also a need for a sterilant gas that can besafely used at high enough concentrations to minimize the time requiredfor sterilization or disinfecting. Also, there is a need for a sterilantgas that does not significantly alter or destroy the materials and/orobjects being sterilized, such as by altering the molecules of thematerials being sterilized or changing the structural form of the objector material .

SUMMARY OF THE INVENTION

The present invention provides a method to generate and use one or moreoxides of nitrogen for the purpose of sterilization and disinfecting.These oxides of nitrogen may include: nitric oxide, nitrogen dioxide,dinitrogen tetroxide or additional oxides of nitrogen individually or incombination. By using compounds that generate nitric oxide onacidification, and combining the nitric oxide with ambient air within anexemplary device, the method generates both water soluble and lipidsoluble oxides of nitrogen each of which possess anti-microbialproperties on their own. In addition, the mixture of gases generated inthe present invention has lower oxidation potential than other sterilantgases, making them safer to handle. Furthermore, the mixture of gaseslacks the potential for explosive hazard possessed by many currentlyfavored sterilant gases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a sterilization device having a sterilization chamber (SC)12, a gas generation chamber (GGC) 14, and a connecting tube 16 having asafety valve 18. The SC 12 has a closure 20, a connecting port 15, andan exhaust port 22 that attaches to exhaust tubing 29. An exhaust valve23 is attached to the exhaust tubing 29. The GGC 14 contains thecomposition capable of generating a sterilant gas (sterilantgas-generating composition) 24. The GGC 14 has a fitting 17 to whichconnecting tubing 16 is attached, and a filling port 21 for addingliquids.

FIG. 2 is a drawing of another embodiment of the Sterilization Chamber12 that has a flap closure 30 for opening or sealing the SC 12.

FIG. 3 is a schematic drawing of a sterilization device 100 of thepresent invention that is comprised of a hard casing with internal gaspumping and scrubbing functions.

The foregoing features of the invention are more readily understood byreference to the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and devices that generate or usenitric oxide, along with other oxides of nitrogen, to sterilize ordisinfect instruments, devices, materials, tools and equipment that mustbe sterile, typically for medical applications. The use of nitric oxidealone, or in combination with oxides of nitrogen that form incombination with air, as a disinfectant and sterilant gas mixture hasseveral advantages over other gases. Neither nitric oxide nor otheroxides of nitrogen are explosive at high concentrations. In addition,because nitric oxide and other oxides of nitrogen have a weakeroxidizing potential than peroxides and ozone, they allow for a broaderlist of materials that can be sterilized. Another advantage to usingnitric oxide and/or other nitrogen oxides is that their density iscloser to that of air, and thus do not settle to the bottom of a closedcompartment when mixed with air, as would chlorine dioxide, which isgreater than twice the density of air.

Generating a mixture of the oxides of nitrogen can have additionaladvantages over pure nitric oxide and other single entity sterilizationgases. Nitric oxide is very lipid soluble and has the ability to disruptthe lipid membranes of microorganisms. Furthermore nitric oxide mayinactivate thioproteins thereby disrupting the functional proteins ofmicrobes. Nitrogen dioxide is more water soluble than nitric oxide.Finally, nitric oxide and nitrogen dioxide are extremely effectivedisruptors of DNA, causing strand breaks and other damage leading to aninability for the cell to function.

A mixture of nitric oxide and air will react, resulting in a mixturecontaining many different oxides of nitrogen. Specifically, the additionof NO to air, or air to NO, results in the formation of NO₂ when NOreacts with the oxygen in air. The concentration of each nitrogen-oxidespecies that is present in a mixture will vary with temperature,pressure, and initial concentration of the nitric oxide.

Definitions

As used herein, the term “gas” or “gases” means any matter that is notin the solid state or liquid state, but rather, has relatively lowdensity and viscosity, expands and contracts greatly with changes inpressure and temperature, diffuses readily and has the tendency tobecome distributed uniformly throughout any container

As used herein, the term “nitric oxide” or “NO” means the NO freeradical or NO. As used herein, the term NO_(x) is an abbreviation fornitrogen oxides or the oxides of nitrogen, which are the oxides formedby nitrogen in which nitrogen exhibits each of its positive oxidationnumbers from +1 to +5. As used herein, the terms “nitrogen oxides” and‘oxides of nitrogen’ and ‘NO_(x)’ mean a gas having one or more of thefollowing gases, all of which contain nitrogen and oxygen in varyingamounts: nitric oxide (NO) nitrogen dioxide (NO₂), nitrogen trioxide(NO₃), dinitrogen trioxide (N₂O₃), dinitrogen tetroxide (N₂O₄),dinitrogen pentoxide (N₂O₅) and nitrous oxide (N₂O). Examples ofpreferred sterilant gases include, but are not limited to NO, NO₂, NO₃,N₂O₃, N₂O₄, N₂O₅. N₂O and mixtures thereof. Examples of the mostpreferred sterilant gases are NO, NO₂, N₂O₄ and mixtures thereof

As used herein, the term “NO-generating” compound or composition means acompound or composition capable of producing or releasing NO, NO₂, andNO_(x). As used herein, the term “sterilant gas-generating” compound orcomposition means a compound or composition capable of producing orreleasing a sterilant gas. An NO-generating compound is one type ofsterilant gas-generating compound. The preferred sterilantgas-generating compounds used in the systems, devices and methods of thepresent invention are carbon-based diazeniumdiolate compounds thatgenerate at least 1 mole of NO per mole of compound.

As used herein, the term “sterilization chamber” means any gas tightchamber of any size, whether comprised of hard or soft materials, whereitems to be sterilized or decontaminated can be contained. Preferably,the sterilization chamber is capable of (i) maintaining a vacuum; (ii)receiving a sterilizing gas; and (iii) receiving air. Sterilization is ahigh-level of decontamination that destroys all microbial life,including highly resistant bacterial endospores. Disinfection in anintermediate-level of decontamination, which eliminates virtually allpathogenic microorganisms, with the exception of bacterial spores. Asused herein, the terms “sterilize” “sterilizing” and “sterilization”mean the killing or removal of all microorganisms in a material or on anobject. When a material or object is “sterilized” or “sterile” there areno living organisms in or on a material or object. Since sterilizationeliminates all microorganisms, including endospores, a method, systemand/or device that sterilizes a material or object, therefore, alsodisinfects and decontaminates the material or object. As used herein,the term “object” refers not to a feature of the invention, but ratherto the article or material being acted upon to be sterilized and/ordecontaminated by the disclosed sterilizing methods, systems anddevices. The term “object” can also include a material to be sterilized,no matter the physical form. An object may include, for example, withoutlimitation, a medical device or medical instrument or any other articleor combination of articles for which sterilization is desired. An objectmay have a wide variety of shapes and sizes and may be made from avariety of materials (e.g., without limitation, metal, plastic, glass).

As used herein, the term “gas generation chamber” means any container,of any size or composition, which may be used to contain a gas and/or agas-generating compound. Preferably, the gas generating chamber is madeof a material that is impermeable to liquid and impermeable to gas. Asused herein, the term “microbe” means any bacteria, virus, fungi,parasite, mycobacterium a or the like. As used herein, the term“scrubbing” means the removal or conversion of toxic oxides of nitrogenfrom the exhaust stream of the sterilization device,

As used herein, the term “medical device” means any instrument,apparatus, implement, machine, appliance, contrivance, implant, or othersimilar or related article, including any component, part, which isintended for use in the cure, mitigation, treatment, or prevention ofdisease, of a human or animal, or intended to affect the structure orany function of the body of a human or animal; and, which is intended tobe inserted, in whole or in part, into intact tissues of a human oranimal. As used herein, the term “implant” or “implantable” means anymaterial or object inserted or grafted into intact tissues of a mammal.

As used herein, the term “impermeable” means a substance, material orobject that prohibits over 95% of any liquid or gas from passing ordiffusing through it, for at least one hour. As used herein, the term“permeable” means a substance, material or object that allows thepassage of gases and/or liquid through it.

The sterilization system and method of the present invention utilizesone or more oxides of nitrogen (individually or in combination) tosterilize a wide variety of devices, instruments, materials, human andanimal tissues, drugs, biologicals, and a variety of medically relevantmaterials.

An additional sterilization system and method of the present inventionemploys compounds that release a sterilant gas, preferably nitric oxide,upon acidification. The system and method of the present inventiongenerates nitric oxide that is used, typically as a mixture of watersoluble and lipid soluble nitrogen oxide gases, to sterilize a widevariety of devices, instruments, materials, human and animal tissues,drugs, biologicals, and a variety of medically relevant materials. Inone embodiment of the present invention, the object to be sterilized ismade of a material that is used in health related products. Examples ofhealth related products are, without limitation, all types of surgicalinstruments; cardiac surgery products; cardiac implants; cardiovascularstents; vascular implants; orthopedic surgery products such as surgicalinstruments, bone graft, bone scaffold; orthopedic implants; dentalsurgery products; dental implants; gastrointestinal implants, urinarytract implants; wound healing products; tissue engineering products. Inanother embodiment of the present invention, the tissue engineeringproduct is a protein.

Typically, an object that is a medical device contains one or morematerials, such metals, a non-metals, a polymers or plastics, anelastomers, as well as biologically derived materials. Preferred metalsused in medical devices are stainless steel, aluminum, nitinol, cobaltchrome, and titanium. Examples of nonmetals are glass, silica, andceramic.

In another embodiment of the present invention, the object to besterilized is made of a material that is a polymer such as a polyesterbioresorbable polymer, for example, without limitation, Poly(L-lactide),Poly(DL-Lactide), 50/50 Poly(DL-lactide-co-glycolide),Poly(e-caprolactone), mixtures thereof. Preferably, the material is abioresorbable polymer capable of being used as an implant material andfor drug delivery. Preferred polymers used in medical devices arepolyacetal, polyurethane, polyester, polytetrafluoroethylene,polyethylene, polymethylmethacrylate, polyhydroxyethyl methacrylate,polyvinyl alcohol, polypropylene, polymethylpentene, polyetherketone,polyphenylene oxide, polyvinyl chloride, polycarbonate, polysulfone,acrylonitrile-butadiene-styrene, polyetherimide, polyvinylidenefluoride, and copolymers and combinations thereof. Other materials foundin medical devices are polysiloxane, fluorinated polysiloxane,ethylene-propylene rubber, fluoroelastomer and combinations thereof.Examples of biologically derived materials used in medical devicesinclude, without limitation, polylactic acid, polyglycolic acid,polycaprolactone, polyparadioxanone, polytrimethylene carbonate andtheir copolymers, collagen, elastin, chitin, coral, hyaluronic acid,bone and combinations thereof.

Certain types of medical devices and implants include a bioactivecoating and/or biocompatible coating, examples of which are, withoutlimitation, infection resistance coating, antimicrobial coating, drugrelease coating, anti-thrombogenic coating, lubricious coating, heparincoating, phophoryl choline coating, urokinase coating, rapamycincoating, and combinations thereof. The bioactive coating can be ahydrophilic or hydrophobic coating. Further examples of bioactivecoatings and polymers include, but are not limited to polyvinylpyrrolidone, polyethylene glycol, polypropylene glycol, polyethyleneglycol-co-propylene glycol, polyethylene glycol acrylate, polyethyleneglycol diacrylate, polyethylene glycol methacrylate, polyethylene glycoldimethacrylate, polyethylene oxide, polyvinyl alcohol, polyvinylalcohol-co-vinylacetate, polyhydroxyethyl methacrylate, andpolyhyaluronic acid, and hydrophilically substituted derivatives,monomers, unsaturated pre-polymers, and uncrosslinked polymers withdouble bonds thereof. Addition bioactive coatings and polymers arepolytetrafluoroethylene, polyethylene, polypropylene, poly(ethyleneterephthalate), polyester, polyamides, polyarylates, polycarbonate,polystyrene, polysulfone, polyethers, polyacrylates, polymethacrylates,poly(2-hydroxyethyl methacrylate), polyurethanes, poly(siloxane)s,silicones, poly(vinyl chloride), fluorinated elastomers, syntheticrubbers, poly(phenylene oxide), polyetherketones,acrylonitrile-butadiene-styrene rubbers, poyetherimides, andhydrophobically substituted derivatives thereof and their precursormonomers.

In another embodiment of the present invention, the object to besterilized is made of a material that is a bioabsorbable polymer or adrug-bearing or drug-eluting polymer or a mixtures thereof. In apreferred embodiment of the present invention, the object to besterilized is an implant.

A preferred embodiment of the system and method of the present inventiongenerates the gases at the point-of-use. Such point-of-use methods,systems and devices eliminate the need for heavy tanks of potentiallyhazardous gases or expensive on-site gas generation plants. Thepoint-of-use gas generation employed in the system and method of thepresent invention can be functional without requiring electricity, whichallows the method to be adapted for portable embodiments forsterilization, disinfecting, and decontamination in austere environmentssuch as combat areas, refugee camps, etc. In one aspect, the presentinvention describes a method to generate a mixture of nitrogen oxidesfor sterilization and disinfecting purposes. The method requires anapparatus that integrates the gas generation and delivery method. Theapparatus used in the process may have many potential embodiments.

In a preferred embodiment of the system or device of the presentinvention, a sterilization chamber is used, along with a source of thesterilant gas comprised of one or more oxides of nitrogen. Thesterilization chamber may be in fluid connectivity with the source ofthe sterilant gas; alternatively, the source of the sterilant gas can bewithin the sterilization chamber. One preferred embodiment includes agas generation chamber in fluid connectivity with a sterilizationchamber. Another preferred embodiment has the gas generation chambercontained within the sterilization chamber.

Also preferred, are embodiments of the system and method of the presentinvention that produces a mixture of nitrogen oxides having lessoxidative potential than commonly used sterilant gases, including ozoneand hydrogen peroxide. An additional advantage is that the mixture ofnitrogen oxides produced has much less explosive potential than thecommonly used sterilant gases, including ethylene oxide, hydrogenperoxide, and chlorine dioxide. This allows the use of greaterconcentrations of the gaseous mixture the system and method of thepresent invention thereby allowing less exposure time in thesterilization cycle as known to those skilled in the art.

Yet another advantage is that the method of the present invention is thegeneration of multiple chemical species with different chemicalproperties for the purpose of sterilization and disinfecting. Thoseskilled in the art understand that multiple mechanisms of cell killingor deactivation are often preferred over single mechanisms of action.Antimicrobial agents with different mechanisms of action are oftensynergistic, producing a greater effect than would be expected by simplyadding the effects from each agent together. The same principle isapplied to microbial resistance, where multiple, distinctly actingagents are used for treatment.

In one preferred embodiment of the method and system of the presentinvention, NO gas is generated using the class of nitric oxide donorsknown as diazeniumdiolates. These compounds spontaneously release NO insolution, with rates that are proportional to the acidity of thesolution. Highly acidic conditions can be used to generate NO in themethod of the present invention, generate NO gas rapidly (completetheoretic release of NO in <30 sec).

A preferred embodiment of the method and system of the present inventionuses carbon-based diazeniumdiolates rather than nitrogen-basedcompounds. Carbon-based diazeniumdiolates are preferred becausenitrogen-based compounds are able to form highly carcinogenicnitrosamine species, as described by Parzuchowski et al., J Am Chem. Soc124: 12182-91 (2002). Also preferred are carbon-based diazeniumdiolatecompounds that release large amounts of NO such as but not limited tothose described in U.S. Provisional Pat. Appl. 60/542,298, (whichproduces greater amounts of nitric oxide per mole of compound, ascompared to the compounds disclosed in U.S Pat. No. 6,232,336) and U.S.Provisional Pat, Appl. 60/542,298, filed Feb. 9, 2004, which isincorporated herein by reference in its entirety. Another embodiment ofthe methods and devices of the present invention employs a sterilantgas-generating composition that includes a nitrogen-baseddiazeniumdiolate compound.

In a preferred embodiment of the methods and devices of the presentinvention that employ a sterilant gas-generating composition thatincludes a carbon-based diazeniumdiolate compound, the carbon-baseddiazeniumdiolate compound produces quantities of NO that are greaterthan 1 mole of NO per mole of the carbon-based diazeniumdiolatecompound. In yet another embodiment of the methods and devices of thepresent invention, the carbon-based diazeniumdiolate has a carbonbearing a diazeniumdiolate group, wherein the carbon does not comprisepart of an imidiatc, thioimidate, amidine or enamine.

In yet another embodiment of the methods and devices of the presentinvention, the carbon-based diazeniumdiolate compound has the formula:

R³—C(R¹)_(x)(N₂O₂R²)_(y)

wherein x is an integer from 0 to 2 and y is an integer from 1 to 3 andthe sum of x plus y equals 3;

wherein R¹ is not an imidiate, thioimidate, amidine or enamine;

wherein R² is selected from the group consisting of a countercation anda protecting group on the terminal oxygen; and

wherein R³ is a phenyl group.

In yet another embodiment of the methods and devices of the presentinvention, the carbon-based diazeniumdiolate compound has the formula:

wherein R¹ is not an imidiate, thioimidate, amidine or enamine;

wherein R² is selected from the group consisting of a countercation anda protecting group on the terminal oxygen; and

wherein R³ is a phenyl. In a preferred embodiment of the invention, R¹is selected from the group consisting of an electron withdrawing group,a nitro group, an ether, a thioether, and a non-enamine amine;

wherein the R² substituent is selected from the group consisting ofaliphatic, aromatic, and non-aromatic cyclic groups; and

wherein the R³ substituent is selected from the group consisting ofmono- or di-substituted amino, unsubstituted amino, ammonium, alkoxy,acetoxy, aryloxy, acetamide, aldehyde, benzyl, cyano, nitro, thio,sulfonic, vinyl, carboxyl, nitroso, trihalosilane, trialkylsilane,trialkylsiloxane, trialkoxysilane, diazeniumdiolate, hydroxyl, halogen,trihalomethyl, ketone, benzyl, and alkylthio. The countercation isselected from the group consisting of an ammonium and other quaternaryamines; and further wherein the protecting group is selected from thegroup consisting of aryl, sulfonyl, glycosyl, acyl, alkyl and olefinicgroups.

One NO-generating compound that may be used in the method and system ofthe present invention, though with caution, is sodium nitroprussidebecause of its concurrent formation of cyanide in the gas generationchamber. The formation of cyanide represents a human health hazard andcreates a disposal safety issue for the gas generation chamber.Nitrosothiols may also be used to generate NO in the current invention,however nitrosothiols have the tendency to reform after they havereleased NO, thus creating a chemical sink for NO and making the releaseof NO unpredictable. A sterilant gas-generating composition of thepresent invention, therefore, may include a nitrogen-baseddiazeniumdiolate compound, such as a nitrosothiol, S-nitrosoglutathione,sodium nitroprusside, molsidomine, an iron-sulfur nitrosyl, Roussin'sblack salt, and mixtures thereof.

A most preferred embodiment of the system and method of the presentinvention, the NO-releasing compound employed is a carbon-baseddiazeniumdiolate compound. Carbon-based diazeniumdiolate moleculesrelease a greater amount of nitric oxide and do not form nitrosamines.Preferably, the carbon-based diazeniumdiolate compound produces greaterquantities of NO per mole. Preferably, a C-based diazeniumdiolatecompound that is capable of producing at least one mole of NO per moleof diazeniumdiolate is used as the sterilant gas generating compound isused in the system and method of the present invention. Such acarbon-based diazeniumdiolate is described in U.S. provisional patentapplication 60/542,298 entitled “Nitric Oxide-Releasing Molecules” filedFeb. 9, 2004; the entirety of which is hereby incorporated by reference.

The system and method of the present invention preferably uses a C-baseddiazeniumdiolate compound that does not result in the formation ofcarcinogenic nitrosamines when acidified. Another advantage of using aC-based diazeniumdiolate compound as the preferred NO-releasing compoundis that it releases a greater quantity of NO per mole of NO-releasingcompound. For example, nitrogen-based diazeniumdiolates andnitrosothiols produce lower quantities of NO per mole compound whencompared to the carbon-based diazeniumdiolate compounds. Also, the useof a C-based diazeniumdiolate compound as the preferred NO-releasingcompound allows the use of an acid to release NO rather than the coppersolution required for release of NO from nitrosothiols. Yet anotheradvantage of the method and system of the present invention is that ithas a reduced environmental impact as compared to a method requiring asolution containing copper ions.

The nitric oxide generating compounds utilized in the system and methodof the present invention provide several advantageous elements to thepresent invention. One advantage is that nitric oxide has a high degreeof lipid solubility, making it toxic to almost all microbes, which havelipid membranes (the exception is non-enveloped viruses).

Nitrogen dioxide, and other oxides of nitrogen such as dinitrogentetroxide, are more water soluble than nitric oxide. These, andespecially nitrogen dioxide, are highly damaging to DNA, resulting innitrosation and deamination of DNA bases and single and double strandbreaks. Damage to DNA is a powerful killing mechanism. Combined, themixture of gases in the present invention provides a multi-prongedattack of microbes through a variety of possible mechanisms of action.The antimicrobial benefits of a method that uses multiple mechanisms ofaction, as discussed above.

Yet another advantage to the system and method of the present inventionis that it can permit the formation of small amounts of nitrous acid andnitric acid in the water that attaches to solids in humid environments,which can enhance the antimicrobial properties of the present invention.

Another embodiment of the system and method of the present inventionuses a gas generating chamber that is a pressurized or non-pressurizedcylinder containing one or more oxides of nitrogen. Though thisembodiment sacrifices portability, it is useful in large scaledecontaminations, such as military or other very large equipment. Theone or more oxides of nitrogen may be stored at high concentrationswithin the cylinder. Although this embodiment is less desirable due tothe hazard and added costs and paperwork involved with shipping ofconcentrated pressurized gases. A more preferred method would be todilute the concentration of the one or more oxides of nitrogen withinthe cylinder to a desired concentration by mixing with nitrogen or otherinert gas including, but not limited to argon, helium, and neon. The gasor gas mixture can be delivered to the sterilization chamber through ametered regulator in fluid connectivity with the sterilization chamber,or other gas delivery method known to one skilled in the art. Anotherembodiment includes computer or microprocessor means to control thedelivery of sterilant gas from the gas cylinder.

In embodiments of the present invention where the NO-releasing entity isactivated by acid, any acid can be used to generate NO. In oneembodiment of the present invention the NO donors are activated by theaddition of a liquid activator that is an aqueous acid, as described inExample 1. The liquid activator may be, for example without limitation,water, an acid, and mixtures of water and acids. Due to theinconvenience of handling and transporting aqueous acids, powdered acidsthat are activated by water are preferred. While any powdered acid wouldbe acceptable, powdered acids with low pKa are preferred because thepreferred method is to rapidly generate the NO, and low pKa acids aremore effective. These low pKa acids include but are not limited tooxalic and maleic acids. Generally, up to ten-fold molar excess ofpowdered acid is used, however lower molar ratios may also beacceptable.

A preferred embodiment of the system and method of the present inventionincludes a gas generation chamber containing both a carbon-baseddiazeniumdiolate and a powdered acid, whereby the gas generation chamberincludes a rapidly sealing opening that allows the addition of a liquid,preferably water, and is in fluid connectivity with the sterilizationchamber so that gas generated upon activation of the carbon-baseddiazeniumdiolate is transported into the sterilization chamber.Additional connections and/or ports may be included for such purposes asto apply a vacuum, if necessary, to release NO gas from the chamber.Preferably, the NO gas is released into a reusable NO scrubbing system.Preferred methods and devices of the present invention include thescrubbing of the sterilant gas after the object is sterilized.

A desiccant may be included in the gas generation chamber to reducemoisture during manufacture, shipping, and storage of the gas generationchamber. Examples of desiccants may include but not be limited tomolecular sieves, silica gels, and other methods known to one skilled inthe art. Care should be taken that the amount of desiccant does notprevent the generation of NO on the addition of water.

One skilled in the art can apply The Ideal Gas Law, the moles of NOreleased from the various NO-releasing compounds, the molecular weightof the compound in question and derive the weight of the compoundnecessary in the gas generation chamber to achieve a desired percent ofNO added to any specified volume that comprises the sterilizationchamber. For example, 1.956 grams of an NO-releasing compound thatgenerates 2 moles of NO per mole of compound having a molecular weightof 163 gms/mole is used to produce 0.0225 moles of NO and provide aconcentration of 50% NO in a one liter volume. This allows the user tocontrol the amount of NO added for various sterilization applications.For example, medical practitioners may desire a more rapid sterilizationcycle, requiring higher concentrations of added NO. Those users who aremore concerned with portability may be less sensitive to speed and costof the process. Longer sterilization cycles may require less of theNO-releasing compound, i.e., less NO added. Thus, the device 100 andprocess offer the flexibility to provide potential end users withoptions regarding cost, speed, portability, and other utilizationparameters.

In one embodiment of the present invention, a lightweight, portabledevice employing chemically generated NO as a rapid, effective sterilantwhich requires no electrical power so that it can be used in austereenvironments.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows the device 10 in its most simplistic form. The device 10 iscomprised of the subcomponents that include the sterilization chamber(SC) 12, the gas generation chamber (GGC) 14, a connecting tube 16 thatallows gas to flow from the GGC 14 to the SC 12, and a safety valve 18,along the length of the connecting tube 16 that separates the GGC 14from the SC 12. The SC 12 has a closure 20, a connecting port 15, and anexhaust port 22 that attaches to exhaust tubing 29. An exhaust valve 23is attached to the exhaust tubing 29. The GGC 14 contains the powderedsterilant gas-generation composition or compound 24, which is describedin detail below. The GGC 14 is further comprised of a female lurefitting 17 to which the connecting tubing 16 is attached, and a fillingport 21 for addition of liquids. Each subcomponent comprising the device10 is described in greater detail below.

FIG. 1 also details the sterilization chamber (SC) 12. The SC 12includes a physical container 13 comprised of a plastic, a closure 20,that is gas impermeable and allows re-opening and re-sealing of the SC12 for loading and unloading of the materials to be sterilized, aconnecting port 15 that allows a gas-tight seal with connecting tube 16,and an exhaust port 22 that allows the removal of the gaseous sterilantfrom the SC 12 prior to removal of the sterilized materials. The SC 12can be comprised of any plastic material that can contain a lowmolecular weight gas for up to 45 minutes, Due to the short duration ofthe period during which it is necessary to contain the gas, semi-gaspermeable materials may be used to construct the SC 12, allowing foroptimization of weight, toughness, and cost parameters for eachindividual application.

Plastics used for the physical container 13 of the SC 12 may includehighly chemical resistant polymers, such as but not limited toStyrene-ethylene-butylene modified block copolymer with silicone oil(for example, C-FLEX tubing), a fluoropolymer made from Halar resin (forexample, Chemfluor 367), Ethylene Propylene Diene Monomer (EPDM),Ethylene TetrafluoroEthylene (ETFE), Polyvinylidene Fluoride (forexample, Kynar), fluoropolymers (for example, MFA), polyketones orpolyetheretherketones (PEEK), perfluoroalkoxy fluorocarbon (PFA),fluoroethylene-propylene (FEP), polyimide, and polyvinylchloride (PVC).The closure 20 may be located at a variety of locations in the SC,preferably, at a point on the SC 12 opposite from the connecting port 15and the exhaust port 22, or towards either side of the SC 12. Theclosure 20 is preferably constructed from polyethylene. One preferredclosure is one having interlocking linear fasteners that are resistantto pressure breaches, such as the U-MAXIGRIP (Illinois Tool Works, Inc.Glenview, Ill.). While many interlocking linear fasteners are available,this model is particularly desirable due to its resistance to pressurebreaches.

An alternate embodiment for the SC 12 is shown in FIG. 2, in which theSC 12 uses a flap closure 30 that is a C-shaped track of interlockingplastic hooks and ribs, sealed or opened by using a hinged zipper-liketab 31 and guide slot 32 that separates or compresses the interlockingcomponents, resulting in the opening or sealing of the SC 12. The flapclosure 30 is positioned 1 to 2 cm from the perimeter of the SC 12 overthree contiguous sides of the SC 12, allowing for a flap of plastic fromthe SC 12 to be pulled back for easy loading and unloading of the SC 12.

The connection port 15 allows for a gas-tight connection between theSC12 and the connecting tube 16. A preferred embodiment includes afemale lure fitting 25 at the top of the connection port 15 whereby theend of the connecting tube 16 can be comprised of a male lure fitting 27or a tapered shaft designed to snugly fit the female lure fitting 25.Alterations in the configuration are well within the skill of the art,such as having the lure fitting at the top of the connection port 15 bea male lure fitting and having the end of the connecting tube 16 be afemale lure fitting.

In one embodiment, the exhaust port 22 is a plastic flange, that is acontiguous unit with the exhaust tube that flanges out from the SC 12and tapers into a length of exhaust tubing 29. Exhaust tubing 29 isfitted with a valve 23 that, when closed, seals the SC 12 from theambient air. In a preferred embodiment, the value 23 is aroller-activated compression valve is many possible embodiments formeans to seal the exhaust tubing 29, known to those of skill in the art.

The connecting tube may be made of any flexible plastic that isrelatively resistant to chemicals. Preferred plastic materials include,but are not limited to, C-FLEX, Chemfluor 367, EPDM, ETFE, Kynar, MFA,PEEK, PFA, FEP, polyimide, and PVC. The length of the connecting tubeshould be sufficient to allow the user to freely manipulate each chamberwithout disturbing the other chamber. Typically, a length of 20 to 30inches of connecting tube 16 is preferred, however lengths outside therange of 20 to 30 inches are also functional. At either end of theconnecting tube 16 there is a male lure fitting. Alternatively, there isa tapered hard plastic tip that can be inserted in the female lurefitting to insure a fluid-tight seal.

A broad range of safety valves 18 may be used to separate the GGC 14from the SC 12 including, but not limited to, crimp valves,roller-activated compression valves, and the like. Any valve that canseal the flow of fluid from the GGC 14 to the SC 12. A preferredembodiment of the present invention employs an air-venting/vacuumbreaking valve because it is self-activated.

Gas Generation Chamber (GGC) and Gas Generation Compound

The GGC 14 includes a container 19 that can be comprised of a variety ofplastics that are chemically resistant. These may include but are not belimited to C-FLEX, Chemfluor 367, EPDM, ETFE, Kynar, MFA, PEEK, PFA,FEP, polyimide, PVC. In a preferred embodiment, the container iscomprised of PFTE and/or polyolefins. The GGC 14 includes a female lurefitting 17 integrated for attachment of the GGC 14 to the connectingtube 16, which allows a contiguous flow of fluid from inside the GGC 14to the SC 12. Preferably, the filling port 21 of the GGC 14 is a large,capped opening, which has a threaded rim protruding at least 0.5 cmabove the wall of the GGC to allow easy grasping and capping.

Another embodiment of the present invention is presented schematicallyin FIG. 3. The sterilization device 100 includes a hard case withinternal gas pumping and scrubbing means that is attached to a sealablegas generation chamber 102. The device 100 is in fluid connectivity witha gas generation chamber 102, through a sealable port 103. In apreferred embodiment, the sealable port 103 may be comprised of a doubleshut off quick disconnect coupling (Colder Products St. Paul, Minn.)where the tubing 104 from the gas generation chamber 102 has the male offemale coupling and the sealable port 103 is comprised of thecomplimentary coupling. The advantage of the double shut off feature isthat disconnection is possible without opening either the gas generationchamber 102 or the sterilization chamber 101 to the local environment.Thus, the sterilant gases are contained within the sterilization chamber101, so that any residual gas from the gas generation chamber 102remains contained until the scrubbing step.

The device 100 has a compartment that is comprised of an electronic orhand operated pump 105 that can be in fluid connectivity to thesterilization chamber 101, or not, depending on the position of anintake valve 106. The intake valve 106 may be manually operated, ormicroprocessor 110 controlled. The intake valve 106 allows the pump 105to remove gas contained in the sterilization chamber 101 and the gasgeneration chamber 102 if it is in fluid connectivity with thesterilization chamber 101 at the time of pump activation. The gas ispumped through a scrubbing system 107 that deactivates and removes thegases from the exhaust stream. The compartment that comprises the innerlumens of the pump 105 and the scrubbing system 107 may or may not befluid connectivity with the sterilization chamber 101 depending on theposition of the intake valve 106. The device 100 is designed so thatafter completion of the sterilization cycle, activation of the valvesand pump 105 draws gas from the sterilization chamber 101 through theintake valve 106 into the scrubbing system 107, through an exhaust valve108 that directs the flow of gas out of the device 100, or back into thesterilization chamber 101 for the purpose of cycling the gas through thescrubbing system 107 for an additional period of time in order to reducethe levels of gas to OSHA or other regulating agency standards orguidelines. During such a recycling of gas, the gas that re-enters thesterilization chamber passes through a sterile air filter 109 to insurethat any potential microbial contaminants picked up by the gas stream(in the pump 105, scrubbing system 107, and the necessary tubing tomaintain fluid connectivity between the sterilization chamber 101 andthese elements) does not enter the sterilization chamber 101 during thegas recycling process.

There are certain risks inherent with gaseous NO that requirespecialized delivery methods and handling procedures. Exposure to highconcentrations of NO is hazardous. The Occupational Health and SafetyAdministration (OSHA) has set the current level of NO that poses anImmediate Danger to Life and Health at 100 parts per million (ppm) for amaximum of thirty minutes before the effects of exposure would pose athreat to health or life. OSHA has also set the levels of NO in theworkplace at 25 ppm time weighted average for eight hours. Because ofthe dangers of potentially lethal doses of NO, any device or deliverysystem must include features to prevent the leakage of NO into thesurrounding environment in a manner and at levels that may raise a riskthat the leaked NO might be inhaled or otherwise applied to subjectsthat may be harmed by such exposure. The formation of nitrogen dioxidealso represents a severe health hazard. OSHA limits for NO₂ are 1 ppmtime weighted average over eight hours.

The system of the present inventions limits the user's exposure to thegases. The system and methods of the present invention include a systemthat can remove and/or detoxify the sterilant gases, otherwise known asscrubbing. The method of the present invention preferably includes ascrubbing process that removes and detoxifies these gases, prior toretrieving the sterilized or disinfected materials from thesterilization chamber. The scrubbing process, includes numerous methodsfor removing and detoxifying the NO, NO₂, and NO_(R). Scrubbing systemsand processes may employ an adsorbent to trap NO, and an oxidizer toconvert NO to NO₂. In appropriate conditions, the sterilant gas may beexhausted to the outside environment, where the concentrations of NO,NO₂, and NO_(x) will dissipate easily. The scrubbing process may beachieved using a commercially available scrubbing device, such as theBuchi Analytical B-414 (New Castle, Del.). Ideally, the scrubbing devicereduces the levels of NO, NO₂, and NO_(x) in the exhaust gas to levelsbelow the OSHA LTWA. Also, see, Basile R. Dealing with Nitrogen OxideEmissions. http://www.finishers-management.com/may2002/nox.htm. It isalso preferred that the method act rapidly.

The method of the present invention most preferably does not expose theuser to concentrations of NO, NO₂, and/or NO_(x) that are above the OSHAlimits. In a preferred embodiment, the gases are removed from thechamber prior to opening the chamber. In some instances such as outdooruse, the chamber may be opened without prior removal of gases. In orderto limit the exposure to the sterilant gases, the system and method ofthe present invention include a system that can remove or detoxify thesterilant gases, otherwise known as scrubbing.

Examples 2, 3 and 4 describe embodiments of effective scrubbing systemsthat use Purakol and Purafil Select (Purafil, Doraville, Ga.). Oneskilled in the art will realize that many configurations of a scrubbingsystem for a mixture of oxides of nitrogen can be designed.

In one embodiment of the present invention, the sterilization system islightweight, requires no electrical (including battery) power, and canbe completely self-contained. The core of the system of a re-usable,sealable sterilization chamber, a disposable gas generation chamber, andconnecting tubing. The re-usable sterilization chamber can be loadedwith surgical instruments or other materials to be sterilized, sealed,and connected to the gas generation chamber which is pre-filled withnitric oxide (NO) donors and acidic activators. Water is then added tothe gas generation chamber, the chamber is sealed, and the generated gasflows into the sterilization chamber. A gas permeable, liquidimpermeable valve separates the two chambers to avoid mixing thecontents of the separate chambers. In one embodiment of the presentinvention, the sterilant gas-generating composition is capable ofreleasing sufficient quantities of NO to sterilize the object in aslittle as about 2 seconds to about 30 seconds. Carbon-baseddiazeniumdiolate compounds are capable of releasing sufficientquantities of NO to sterilize the object in as little as about 2 secondsto about 30 seconds. A duration of five minutes can be sufficient forsterilization, although a safety margin of an additional ten minutes isprudent.

EXAMPLE 1 Sterilization with Varying Quantities of Delivered NitricOxide

A blood storage container (Nexell, Irvine, CALIF.; Lifecell PL732plastic tissue culture flask) is used as the sterilization chamber.Strips of stainless steel are dipped in 10⁶ CFU/ml sporulated B.subtilis var. niger (as determined by ABS₅₉₅ standardization curves).The strips are allowed to air dry, placed in the sterilization chamber,and then the sterilization chamber is heat sealed. The sterilizationchamber is evacuated using a syringe and controlling air flow with thesterilization chamber valves. A known quantity of air is added to thevessel using a graduated syringe.

An NO-generating donor compound is placed in a 7 cc gas generationchamber. The gas generation chamber is attached to the storage containerthrough luer lock connectors. The liquid activator, 3.0N HCl is added tothe gas generation chamber and the generated gas is allowed to flow intothe sterilization chamber. After a brief generation period, the gas issealed in the sterilization chamber using a compression valve.

Varying quantities of NO gas, namely 10%, 5%, 2.5% and 1% NO, are testedfor their efficacy in the sterilization chamber. The quantity (%) of NOgas generated and added to the sterilization chamber is calculated fromthe number of moles of NO required to be generated to result in thedesired percentage of NO. This calculation uses the Ideal Gas Law andformula weight of the NO gas-generating compound, which in this Exampleis a diazeniumdiolate NO donor, to determine the mass of NO gasgenerating compound to be used.

All percentages tested, including 1%, are effective at killing 10⁶CFU/ml sporulated B. subtilis var. niger in five minutes, as determinedby culturing of the contaminated steel strips in LB at 37° C. andvigorous shaking for 48 hours, followed by plating onto agar plates.Controls are identically treated with the exception of the addition ofpercentages of nitrogen in place of NO. Control stainless steel stripsexhibited visible growth after 24 hours of incubation under the statedconditions.

EXAMPLE 2 Scrubbing of NO, NO_(x) from a Portable Sterilization Chamber

After the sterilant gas is used in the sterilization chamber, the gas inthe chamber is evacuated to another chamber containing scrubbing media.The evacuated gas is allowed to reside over the scrubbing media.

Two 300 ml PL732 tissue culture bags (Lifecell PL732 plastic tissueculture flask case, Nexell, Irvine, Calif.) are connected to each otherwith tubing. A hose clamp is used to close one bag off from the other.An incision is made in one bag, designated to be the ‘scrubbing’ bag,into which a pre-measured amount of scrubbing media (6.0 to 60 grams ofPurafil Select and Purakol in a 1:1 mixture) is added to the bag. Theincision is then heat sealed. Both bags are evacuated with a syringe.Air (180 cc) is injected into the bag designated to be the sterilizationchamber. Thereafter, 20 cc of NO gas is injected to reach a finalconcentration of 10% NO. The mixture of NO and air is allowed to remainin the sterilization chamber for 5 minutes, Thereafter, the hose clampis removed and the sterilization bag is compressed to push all of the NOgas into the scrubbing bag containing the Purafil Select and Purakol.The hose clamp is then secured. Immediately thereafter, samples (0.1 to1.0 cc) of the atmosphere in the scrubbing bag are taken and injectedinto an NO detector, which measure the concentration of NO in parts perbillion (ppb). Thereafter, 1.0 cc samples of atmosphere in the scrubbingbag are taken at timed intervals and injected into the NO_(x) detector.Results of three successive trials are shown in Table 1. The scrubbingmaterials need not be changed between successive trials.

TABLE 1 Scrubbing of NO_(x) gas Time minutes Trial 1 [NO] ppb Trial 2[NO] ppb Trial 3 [NO] ppb 0 32556 69685 69094 5 686 nd 999 6 nd 1484 Nd10 76 nd 253 12 nd 102 Nd nd = no data

EXAMPLE 3

This example provides a method of scrubbing NO_(x) by flowing the NO_(x)gas through tubing filled with scrubbing media, which is connected to acontainer. Tubing (30 inches of ⅜ inch ID and ½ inch OD silastic tubing)is filled with 13.3 grams of a 1:1 mixture of Purafil Select andPurakol. Some of the media is crushed in this process. Glass wool plugsare inserted in the ends of the tubing. Each end of the tube isconnected to separate plastic tissue culture bag (Lifecell PL732 plastictissue culture flask, Nexell, Irvine, Calif.). One bag includes aninline valve. The bags are evacuated of atmosphere and the valve isclosed. One bag is designated the sterilization chamber, and injectedwith 180 cc of air and 20 cc of NO gas. The gas is allowed to remain inthe sterilization back for five minutes. The valve is then opened andthe gas pushed through the tubing into the receiving bag. A 0.5 ccsample of the atmosphere in the receiving bag is injected into theNO_(x) detector. The results show that the contents of the receiving bagis 30 ppb NO_(x), a concentration well below the OSHA guidelines.

EXAMPLE 4 Scrubbing of NO, NO_(x) from Sterilization Chamber

A sealable case (Pelican Products, Inc., Torrance, Calif.) is modifiedwith additional ports comprised of EFC 12 Series Quick DisconnectCouplings (Colder Products Company, St. Paul, Minn.) and a plastic shelfwith a self-sealing gasket edge which divided the case into upper andlower sections of approximately equal volume. The upper section is thesterilization chamber, which has a volume of 20.3 liters (4.5 in by 19in by 14.5 in). One port into the sterilization chamber is used tointroduce a known amount of NO gas into the sterilization chamber, andoptionally, allowing for a recirculating flow. An exhaust port on theopposite end of the case is in the disconnected (sealed) state for thesteps involving the addition of NO gas and during the 5 minute timeperiod to approximate sterilization cycle time.

The lower chamber stores the pump, microprocessor and electriccomponents if any, the valves, the scrubbing system, the sterile airfilters and, optionally, additional components. The scrubbing system isconnected to the exhaust port and includes tubing having a male end ofthe EFC 12 Series Quick Disconnect Couplings. Distal to the exhaustport, the tubing is connected to a pump (Gast, Benton Harbor, Mich.;Model DOA-P104-AA; 35 lit/minutes flow rate), followed by columns thatcomprise the scrubbing system. One column is filled with Purafil Select(Doraville, Ga.); the other is filled with Purakol (approximately 200 to300 grams of material for each column). NO is injected into the uppersterilization chamber and held for a 5 minute period. After 5 minutes,the scrubbing system is engaged by attaching the male end of the EFC 12Series Quick Disconnect Couplings to the female end of the exhaust port,thus opening the port, and activating the pump. Prior to pumpactivation, the pump exhaust is reconnected to the sterilization chambervia the same port that is used to add NO to the sterilization chamber,using tubing ending with a male end of the EFC 12 Series QuickDisconnect Couplings, and also comprised of a sterile air filter (ACRO50, Pall Corporation, Port Washington, N.Y.). The gas from thesterilization chamber is sampled using a syringe from an in-linesampling vessel fitted with a rubber septum after 1 minute of pumping.The sampled gas is then injected into and quantified by theThermoEnvironmental (Waltham, Mass.) 42C chemilluminescent NO_(x)detector. In addition, NO from the NO storage vessel is injected on themachine as a positive control. The system can recirculate, for example,by adding the gas, disconnecting the gas generation chamber, add tubingfrom the exhaust port, back to the “intake” port where the NO was addedoriginally, and, when the pump is turned on, the gas recycles throughthe system.

One set of experiments is performed in quadruplicate on the device using1% added NO. After one minute of recirculating the gas from the exhaustport back through the intake port (using sterile air filters toeliminate contaminating the sterilization chamber), as described above,sampling the gas content of the sterilization chamber and measuringshowed that virtually all of the NO and NO components are removed. Eachof the four samples barely raised the baseline of the NO detector,resulting in a reading estimated to be approximately 2 ppb, far belowthe OSHA guidelines of 25 ppm for NO and 1 ppm for NO₂.

Experiments are performed using 5% added NO. One liter of air (5%) isremoved from the sealed case prior to addition of 5% NO, so that theexperiment is performed at atmospheric pressure. One liter of NO is thenadded to the sealed sterilization chamber and allowed to remain for 5minutes. The scrubbing system is then activated as described above.After one minute of gas recirculation, samples showed approximately 4ppb for NO and NO_(x), in each experiment, again far below OSHAguidelines. The Purafil Select and Purakol columns are not changed overthe course of these 6 experiments.

EXAMPLE 5

A glass pressure vessel is connected to a scrubbed NO gas tank source.The pressure vessel is purged five times with Argon gas to eliminateatmospheric oxygen (preventing formation of NO₂) and an additionalthree purges of NO are used to ensure a pure NO atmosphere andconsistent results of bactericidal activity. To test the sterilizationmethod,: Bacillus subtillis var niger 9372 is used (after obtaining >80%endospore formation; a standard for ethylene oxide and autoclave teststerilization), as well as organisms commonly found on the epidermis:Staphylococcus aureus (strain 21769) and Staphylococcus epidermides(strain 21977), and the enteric organisms: Klebsiella pneumoniae (strain21991) and Serratia marcesens (strain 21140). This particular Serratiastrain has been found in previous studies to be one of the mostresistant bacteria to the bactericidal effects of NO in culture. See,Raulli R et al., Recent Res Devel Microbiol 6: 177-183, 2002, the entirecontents of which is incorporated by reference herein.

The organisms are cultured overnight in brain heart infusion BHI. Thecultures contain at least 10⁸ CFU/ml based on standardized ABS₅₉₅ curvesfor each organism. Stainless steel strips, 3×1 cm, are dipped in thecultures, and either dried in ambient air first or placed in thepressure vessel still wet from the culture dip. The strips are exposedto NO gas at atmospheric pressure for decreasing time periods startingwith 45 minutes and working back to 5 minutes. Control samples arehandled identically, except the pressure vessel is gassed with nitrogen.

TABLE 2 Results from Pressure Vessel Experiments at Five MinuteSterilization Cycle Bacillus Serratia Staphylococcus KlebsiellaStaphylococcus subtilis marcesens epidermides pneumoniae aureusStainless steel 3/3 Killed 3/3 Killed 3/3 Killed 3/3 Killed 3/3 Killed

The sealed vessel is carefully opened in a laminar flow hood after allthe NO had been purged with Argon. The samples are removed asepticallywith sterilized tongs and placed in culture tubes containing sterile BHImedia. The samples are incubated in a vigorously shaking waterbath at35° C. The samples are observed (digitally photographed) 24 hrs later,placed back in the waterbath and are measured for absorbance 72 hrslater. The controls had >10⁸ CFU/ml after 24 hrs. The results shown inTable 2 are from three separate experiments and the results (3/3)indicate that 3 out of 3 trials showed no bacterial growth.

EXAMPLE 6

Similar to Example 1, a portable system is devised using blood storagecontainers and other laboratory items. In this construct, a bloodstorage container (Nexell, Irvine, Calif.; Lifecell PL732 plastic tissueculture flask) serves as the sterilization chamber. It has multipleports, is easily attached to tubing or other chambers, and is easily cutand heat sealed for insertion and removal of contaminated/sterilizedsamples. The heat seal is strong and holds well at pressure, even though1 ATM of pressure is used experimentally. Two 60 ml syringes connectedto each other and a line of tubing by a three-way stopcock are employedto mix acidic buffer in one syringe with NO-releasing diazeniumdiolatein the other syringe. The tubing is connected to the bloodcontainer/sterilization chamber.

The stopcock is turned so that the acidic buffer can be added to thesyringe containing the diazeniumdiolate. The valve is immediately closedto the buffer syringe and opened to the sterilization chamber. The 300cc sterilization chamber inflates in about 15 seconds. Experiments areperformed as described above, except that the devised system is usedinstead of the pressure vessel.

The test organisms are cultured overnight in BHI. The cultures containedat least 10⁸ CFU/ml (100-fold greater than FDA testing guidelines) basedon standardized ABS₅₉₅ curves for each organism. Stainless steel strips,3×1 cm, are dipped in the cultures, and either dried in ambient airfirst or placed in the pressure vessel still wet from the culture dip.The dried samples are dipped in sterile BHI media before being placed inthe sterilization chamber. This prototype is shown to exhibitbactericidal activity against wet dipped stainless steel stripscontaminated with B. subtilis (endosporulated), B. subtilis(vegetative), S. marcesens, or S. epidermides in 15 minutes and it maybe possible to achieve sterilization in less time.

TABLE 3 Results from Fifteen Minute Sterilization Cycle Bacillussubtilis Bacillus Staphylococcus Serratia (spore) subtilis epidermidesmarcesens Stainless 2/2 2/2 3/3 3/3 steel strip Killed Killed KilledKilled

EXAMPLE 7

In this Example, the sterilization of medically relevant materials, suchas needles and plastic tubing, is tested. Teflon (⅛′ ID), polyethylene(1.77 mm ID), vinyl (0.5 mm ID) tubing and a 30 gauge disposable needleare dipped in a bacterial cocktail containing B. subtilis, S. marcesens,and S. epidermides at about 10⁸ total CFU/ml The samples are placed inthe sterilization chamber and sealed. In each case, the lumen (the inneropen space or cavity within the tubing) of the tubing or the needlecontained at least some visually confirmed inoculum. Table 4 shows theresults from the study. The controls for each material reached at least10⁶ total CFU/ml in 24 hrs as determined by ABS₅₉₅ standardizationcurves.

TABLE 4 Sterilization of Medical Materials Vinyl Polyethylene TeflonDisposable Tubing Tubing Tubing Needle 0.5 mm ID 1.77 mm ID ⅛' ID 30gauge. 15 min 2/2 2/2 2/2 2/2 Sterilization Killed Killed Killed KilledCycle

EXAMPLE 8 Humidity Effects on Live Bacteria

This Example tests several humidifying paradigms and the ability tosterilize through a gas sterilization seal pouch. A cocktail of bacteriais used, grown to about 10⁸ CFU/ml and mixed in equal volume. Stainlesssteel strips are dipped, allowed to dry, and subject to one of threemethods: A, B and C. Method A samples are wrapped in a moist Kimwipe.Method B samples are left dry. Method C samples are dried and sealed ina V. Mueller™ Dual Peel Seal Pouch, which is designed for gas andautoclave sterilizations. Samples from methods B and C are placed in thesterilization chamber with a moist Kimwipe. Placement in the chamber isdone so as to insure maximal separation of the Kimwipe and thesample(s). The chamber is re-sealed carefully so as not to disturb thepositioning of the samples relative to the Kimwipe. Each sample isexposed to a 15 minute sterilization cycle at 1 atmosphere, removedunder sterile conditions, and the samples are placed in BHI media asdescribed above. Experimental samples are exposed to NO gas whilecontrol samples are handled identically, except that the chamber isgassed with nitrogen. The results are shown in Table 5. All controlsreached greater than 10⁶ total CFU/ml in 24 hrs as determined by ABS₅₉₅standardization curves.

TABLE 5 Effect of Moisture on Sealed and Unsealed Dry Samples Method AMethod B Method C Sample Moist Kimwipe Dry Dry, Sealed 15 min KilledKilled Killed Sterilization cycle 2/2 2/2 2/2

This experiment suggests two highly significant findings. One is thatsterilization of samples contaminated with live bacteria do not requireadded moisture from a Kimwipe, since NO sterilized dry samples withinthe chamber. The second is that the sterilization can occur within asealed wrapper, which can preserve the sterility of the instrument afterthe chamber is opened. When this sterilization method employs a gasgenerating compound, it provides a lightweight method that requires noelectrical power and is highly transportable.

EXAMPLE 9 Humidity Effects on Spores

This Example illustrates the effects of humidity on the sterilization ofspores using both NO and NO₂/N₂O₄ as sterilizing gases. Tests areconducted in 300 ml glass vessels. The test procedure is as follows:Half of the vessels are humidified by adding 40 micromilliliters ofwater in the vessel and sealing the vessel with Parafilm. On the insideof the vessels that are prehumidified with 40 ml of water, watercondensate is visible over the course of the tests, indicating a highlevel of humidity in such vessels. The vessels into which no water wasintroduced showed no condensate on the inside wall of the vessel.

The vessels are allowed to stand for 30 minutes, Two Tyvek sachets, eachcontaining a Biological Indicator manufactured by Raven (product number3367771 3-6100ST) are placed in each vessel. The vessels are purged asfollows: The vessel is evacuated to 8″Hg absolute. The Vessel isre-filled with air to atmospheric pressure. Evacuation/filling isrepeated two more times. The vessel is evacuated to 8″Hg absolute.Sterilizing gases are introduced into the vessel. Compressed air (9%Relative Humidity) is added until the vessel reaches atmosphericpressure. The sachets are allowed to remain in the sterilizingenvironments for either five or ten 10 minutes before being removed andthe BI is cultured in 4 milliliters of Tryptic Soy Broth for 14 days at55-60° C.

Results are shown in the following Table 6.

TABLE 6 Nitrogen Sterili- Oxide zation Humid Spore Test Oxide of Concen-Exposure or Dry Condi- Num- Nitrogen tration Time Condi- tion in berUsed Diluent (%) (Minutes) tion 14 Days 1 NO₂/N₂O₄ Air 5 5 Humid Dead 1ANO₂/N₂O₄ Air 5 10 Humid Dead 2 NO₂/N₂O₄ Air 5 5 Dry Alive 2A NO₂/N₂O₄Air 5 10 Dry Alive 3 NO Air 5 5 Humid Dead 3A NO Air 5 10 Humid Dead 4NO Air 5 5 Dry Alive 4A NO Air 5 10 Dry Alive 5 NO₂/N₂O₄ Nitrogen 5 5Humid Dead 5A NO₂/N₂O₄ Nitrogen 5 10 Humid Dead 6 NO₂/N₂O₄ Nitrogen 5 5Dry Alive 6A NO₂/N₂O₄ Nitrogen 5 10 Dry Alive

These tests illustrate the importance of humidity in killing spores. Inadditional sterilization cycle tests, spores are also killed at roomtemperature in humidity levels between 40% and 80% Relative Humidity.

EXAMPLE 10 Testing of Powdered Acids

A preferred sterilant gas-generating composition is comprised of anitrogen-based diazeniumdiolate and oxalic acid. Addition of the oxalicacid in a 10:1 molar ratio with the diazeniumdiolate provides producesthe sterilant gas, NO, from the diazeniumdiolate, filling a bloodstorage container in about 20 sec. This capability eliminates thenecessity to add 3N HCl to the diazeniumdiolate to generate NO, insteadallowing the addition of water to activate the release of NO gas. Theelimination of the need for acid makes the device significantly moreconvenient to ship, store, and use.

A disposable, plastic gas generation chamber that can be pre-filled witha carbon-based diazeniumdiolate (nitrogen-based diazeniumdiolates canpossibly decompose to form carcinogenic nitrosamines) plus an activatingpowdered acid, have a large, capped opening to ease the addition ofwater, and have appropriate attachment lines to transport the gas intothe sterilization chamber. Other utility lines or ports may besubsequently added to pull a vacuum if necessary, and to release NO gasfrom the chamber (through a reusable NO scrubbing system).

A polyolefin material chosen for its flexibility, puncture resistance,light weight, and ease of manufacture. The size is approximately a flat10 inch square. The sterilization chamber's bottom edge has a tabbed“Ziploc” like re-sealable opening, allowing quick and easy loading ofinstruments and re-sealing. After the user places the instruments in thepouch, the top portion of the pouch is sealed with a simple quick motionof the tab, resulting in a complete gas tight seal.

One edge of the pouch sterilization chamber has an embedded tubing portand approximately 10 inches of tubing to provide a connection to the gasgeneration chamber. The end of the delivery tube are “quick disconnect”fittings to facilitate easy connection to the gas generation chamber,and each piece of tubing has compressing roller valves to seal the tube.

The chamber is made of a polyolefin material, be 3.5 inches square, andhas a large hard plastic screw cap protruding from the top side of thecontainer for easy filling of powders and water. The chamber has a lurelock port to allow easy connection to the sterilization chamber.

EXAMPLE 11 NO Gas Generation from Nitrite Metal Salts

Nitric oxide is generated using solid nitrite metal salts (for example,sodium nitrite, potassium nitrite, magnesium nitrite) and reacting witheither a liquid acid solution (for example, sulfuric acid , maleic acid,hydrochloric acid, nitrous acid, nitric acid) or proton donors insolution to form nitric oxide and/or nitrogen dioxide. Nitric oxide mayalso be generated by preparing solutions of nitrite metal salts andreacting the solution with solid acid powders to form nitric oxideand/or nitrogen dioxide. If the metal salt and the solid acid powdersare mixed in powder forms, the addition of water will initiate thereaction of the two powders. Nitric oxide gas is generated utilizingboth nitrite metal solutions and acid solutions. Nitrous acid can beformed during these types of reactions; the nitrous acid can decomposeover time to form nitric oxide and nitrogen dioxide. The NO gas can beused to sterilize Biological Indicators or objects and materials, usingthe method and system of the present invention.

For example, maleic acid and NaNO2 are added to a 20 mL vial; the vialis placed in 1 L jar. Two BI's (Raven Lot #3367552) in closed sachets(tyvek facing toward NO generating system) are placed inside the 1 Ljar. 5 mL of water is added to the powder using a syringe. After 10minutes, the BI's are placed in 4 mL tryptic soy broth for 14 days at55-60° C. Alternatively, the BPs in sachets can be taped to the side ofthe 1 L jar taped so that the tyvek faces the NO source. Also, air maybe removed from jar. A vial containing maleic acid and NaNO2 within thejar can be injected with water using a needle. After 10 minutes, theBI's are placed in 4mL tryptic soy broth for 14 days at 55-60° C.Alternatively, a water soluble capsule can be used to hold a mixture ofpowdered maleic acid and powdered NaNO2, which is then dissolved withwater at the start of the sterilization cycle.

Table 7 illustrates a variety of combinations of sodium nitrite andmaleic acid quantities and ratios that can generate adequate quantitiesof NO gas to sterilize a Biological Indicator in the time indicated.

TABLE 7 Maleic Acid Quantity Amount Exposure (g) NaNO₂ time (min) 1.010.503 10 1.028 0.509 10 1.001 0.5 10 1 0.504 10 1 0.518 10 1.01 0.504 101 0.506 10 1.03 0.374 10 1.01 0.33 10 1.16 0.332 10 1.01 0.255 10 10.109 60 0.2538 0.1258 30 0.257 0.128 30 0.25 0.131 30 0.25 0.125 300.25 0.119 30 0.255 0.128 30 0.223 0.127 20 0.2477 0.1312 20 0.244 0.13210 0.2478 0.1326 10 0.246 0.119 5

EXAMPLE 12 Synthesis of a Soluble, Carbon-Based Diazeniumdiolate

While a variety of nitrogen-based diazeniumdiolates are commerciallyavailable and would work in this application, the ability ofnitrogen-based diazeniumdiolates to form highly carcinogenicnitrosamines limits their use in medical applications (Parzuchowski etal., 2002, cited above). The carbon-based diazeniumdiolates cannot formnitrosamines and can produce up to three times more NO on a molar basisthan nitrogen-based NO donors, By using a carbon-based NO donor, themargin of safety for the product is increased while decreasing the totalweight.

A carbon-based diazeniumdiolates can be produced through the use of abenzylic intermediate. Benzyl methyl ether, PhCH₂OCH₃ (commerciallyavailable from Sigma-Aldrich, St. Louis, Mo.) is one starting material.In a Parr pressure vessel, 3 ml (0.024 moles) of benzyl methyl ether isadded to 30 ml of methanol. To this solution, 11 ml (0.048 moles) of 25%sodium methoxide is added with stirring. Oxygen is removed from theflask by alternating cycles (10) of inert gas pressurization andventing. The solution is then exposed to between 40 and 80 psi NO gas atroom temperature for 1 to 5 days. When no more NO gas is being consumed,the reaction is complete, and the head space is purged of NO gas.Diethyl ether is then added to precipitate out all of the anionicdiazeniumdiolated salts, which may then be filtered and dried. Theproduct, PhC(N₂O₂Na)₂OCH₃ is tested for its ability to release NO usinga chemilluminescent method described below as well as subject tostructure verification by spectrophotometry, elemental analysis, and NMRconfirmation.

An alternate synthetic scheme can be derived from the commerciallyavailable benzyl thiocyanate (PhCH₂SCN, Sigma-Aldrich, St. Louis, Mo.).In a Parr pressure vessel, 3 g (0.020 moles) of benzyl thiocyanate isadded to 30 ml of tetrahydrofuran. To this solution, 40 ml (0.040 moles)of 1.0 M sodium silanolate is added with stirring. Oxygen is removedfrom the flask by alternating cycles (10) of inert gas pressurizationand venting. The solution is then exposed to between 40 and 80 psi NOgas at room temperature for 1 to 5 days. When no more NO gas is beingconsumed, the reaction is complete, and the head space is purged of NOgas. Diethyl ether is then be added to precipitate out all of theanionic diazeniumdiolated salts, which may then be filtered and dried.The product, PhC(N₂O₂Na)₂SCN is tested for it ability to release NOusing a chemilluminescent method described below as well as subject tostructure verification by spectrophotometry, elemental analysis, and NMRconfirmation.

Preferred sterilant gas-generating compounds are these carbon-baseddiazeniumdiolate compounds for generating NO because their NO releaserate under acidic conditions rapid and close to identical. The likelycriteria for choice of NO donor is yield and cost.

EXAMPLE 13 Determination of NO Release from Diazeniumdiolates

The method for determining the NO released from diazeniumdiolates isconducted according to the method of Smith D J et al., J Med Chem39:1148-1156, 1996, the entire contents of which is incorporated herein.Weights of the samples is recorded and placed in 0.1 M phosphate buffer(pH 7.4) and the mixture is allowed to stand open to the air at 25° C.in a water bath. The buffer is then purged with argon gas via a frittedglass tube at the bottom of the vessel, such that the gaseous effluentgas is passed through a chemilluminescent NO_(x) detector calibrated tomeasure NO content. Bubbling is continued until a steady and horizontaltrace is achieved, whereupon the signal is integrated over a span ofseveral minutes. The number of integral units are converted to a valuefor moles of NO by comparisons with integrals obtained for certifiedgaseous standards of NO in helium (MG Industries, Morrisville, Pa.). Therate of NO release over that time increment, calculated by dividing theintegrated signal by the number of minutes the integration is conducted,are plotted versus the total elapsed time since the sample was firstplaced in the buffer.

EXAMPLE 14 Environmental Containment of NO

All experiments involving NO are performed in a certified fume hood. NOis an environmental pollutant and may be harmful to humans atconcentrations higher than 100 ppm. NO contained in synthesis vessels orin sterilization chambers are bled into a container that holds 10-foldthe volume of ambient air for a duration of 5 minutes. This step servesto turn all of the NO into NO₂. The NO₂ from the chamber then passesover a column of NaOH, which effectively scrubs out the NO₂. This is awell characterized method commonly used in industrial processing(Basile, 2002).

EXAMPLE 15 Optimization of Sterilization Cycle at Ambient Temperature

The following parameters are optimized for a sterilization cycle atambient temperature (˜22-24° C.), the cycle duration percent airallowable, humidity, internal pressure (amount of NO), and instrumentcharacteristics (surface area of instruments, types of instruments[i.e., narrow lumens, dead end lumens], use of pre-packaged materials insterilization pouches, salt-crusted instruments, protein crustedinstruments). The biological indicator (BI) organism chosen for testingis sporulated Bacillus subtillis var niger, which is the standardorganism used for Et₂O process validation and is also commonly used forother sterilization process validation. See Hoxey E V et al., J ApplBacteriol 58: 207-214, 1985, which is incorporated herein by referencein its entirety.

B. subtillis var niger 9372 is cultured overnight in Luria Broth (LB)media at 37° C. in a shaking waterbath. This usually results in aculture of greater than 10⁸ CFU/ml. The ABS₅₉₅ is measured for eachovernight culture and compared to a standard curve to determine theapproximate CFU/ml. The density of the cultures is adjusted to 10⁶CFU/ml by dilution with sterile LB. The bacillus is sporulated accordingto the following method. The cultures are centrifuged at 2500 RPM (1000x g, Sorvall GLC-1) for 5 minutes and resuspended in a low nutrient saltmedia as described by Sterlini and Mendelstam, Biochem J. 113:29-37,1969 (1969). The bacteria are washed twice more in this sporulationmedia, and the final pellet suspended in an appropriate amount ofsporulation media to retain a density of 10⁶ CFU/ml. This methodtypically results in greater than 80% endospore formation.

Paired stainless steel strips, Teflon® tubing sections 1″ long, ⅛″ innerdiameter (I.D.), and (polyethylene)terephthalate (PET) strips are usedfor general studies of the Sterilization Cycle Parameters studies. Thesethree materials, stainless steel strips, Teflon tubing and PET stripsare referred to as the “Materials Panel” Items from the Materials Panelare dipped in 10⁶ CFU/ml of the endosporulated bacillus suspension.Paired material samples are handled identically, with the controlmaterials being placed in a sterilization chamber and exposed tonitrogen under the same conditions as the group undergoing sterilizationwith NO gas. After processing, the materials are placed in LB media forincubation at 37° C. in a shaking waterbath for 24 hrs. The culturebroth for control and processed groups are observed visually andphotographed at 24 hrs. An aliquot is removed, serially diluted withsterile LB, and plated onto LB agar plates to determine the CFU/ml. Theculture is incubated for an additional 24 hrs to total 48 hrs, and ifneeded the ABS₅₉₅ is measured at 48 hrs (along with additionalconfirmatory photographs). The inoculated LB agar plates are incubatedfor 37° C. and assessed for colony growth 24 and 48 hrs after plating.

Any plate colonies that arises from materials that have been through thesterilization process is tested to confirm the identity of the bacteriaas B subtillis var niger through morphological, Grain stain and/or othernecessary means. The same confirmatory step applies to any cultures inLB that turn positive. Any tested parameter that results in material(s)that exhibit a B. subtilis var niger positive culture after beingexposed to the sterilization process is considered a parameter beyondthe usable range.

EXAMPLE 16 Assessment of Sterilization Cycle Duration on SterilizationEfficacy

Sterilization of the materials are tested at 5, 10, 20, 40, 80, and 120minutes at room temperature on the Materials Panel. For each processedgroup, a control group is treated identically, with the exception ofusing nitrogen gas instead of NO. The experiments are repeated threetimes, and the criteria for successful sterilization at any particulartime point is 0 CFU/ml in all three trials. One failure (positive B.subtillis var niger culture) in three trials is considered a failure atthat measurement.

EXAMPLE 17 Effect of Ambient Temperature on Sterilization Efficacy

Items from the Materials Panel are dipped in 10⁶ CFU/ml B. subtillis inLB. An appropriate time point is selected using data from the previousexperiment, using the penultimate minimal successful time point. Forexample, if 5 minutes is successful, then 10 minutes is used.Experiments are performed from −10° C. to 50° C. in ten degreeincrements. Should either of the extreme temperatures fail to produce asuccessful outcome, the temperature is increased or decreased by 10° C.and the trial repeated until a successful outcome is reached. Coldexperiments are performed in a calibrated refrigeration unit capable oftemperatures from −20° to 20° C. Beyond 20° C., the experiments areperformed in a standard incubator. The sterilization device componentsare equilibrated to the test temperature for 20 minutes prior to thesterilization process trial. For each processed group, there is acontrol group treated identically, with the exception of using nitrogengas instead of NO. A successful outcome at any temperature point is 0CFU/ml in all three trials. One failure (positive B. subtillis var nigerculture) in three trials is considered a failure at that measurementtherefore setting the limit at the measured parameter,

One possible interdependency would be the relationship between higherambient temperatures and NO gas pressure used in the process. It may bereasonably predicted that higher temperatures may result in an equal orgreater degree of efficacy with less NO gas pressure. This would notnecessarily be a problem. A problem that may surface is the ability tohumidify the sterilization chamber under freezing temperatureconditions. In this case, the inability to humidify the chamber mayimpose a limit on utilization of the process in freezing temperatures.

EXAMPLE 18 Evaluation of Optimal Humidity Conditions in theSterilization Chamber

A manufactured sterilization chamber prototype is altered to allow theinsertion of a hygrometer probe. The probe is sealed inside the chamberusing a non-hardening silicon sealant. A NIST traceable hygrometer(Fisher Scientific) with a range of 2 to 98% relative humidity (RH) isused to measure humidity levels. The calibration of the meter is checkedonce a week using dedicated nitrogen-gassed chambers containing saltbaths mixed to generate RH environments of between 10 to 80%, such as11, 43, and 75%.

The method producing reproducible RH levels in the sterilization chamberis established, and thereafter, items from the Material Panel arecontaminated with B. subtillis, allowed to dry in ambient air, placed inthe sterilization chamber along with an appropriate weight of water(absorbed on swatches) and the sterilization process is tested along thelinear range of RH achievable in increments from 10-80% RH, for example10 to 15% RH, 20%, 30%, 40%, 50%, 60%, 70% and 80% RH, Experimentsperformed at test temperatures other than room temperature firstequilibrate at the test temperature for 20 minutes prior to beginningthe sterilization process. Again, the penultimate minimal effective timepoint is used. A successful outcome at any RH level is 0 CFU/ml in allthree trials. One failure (positive B. subtillis var niger culture) inthree trials is considered a failure at that measurement thereforesetting the limit at the measured parameter.

For example, if the sterilization fails at 0% RH but is effective at15%, then additional experiments are conducted to identify the precise %RH between 0 and 15% to optimize the range of effective conditions forthe humidification and/or desiccation needed in the sterilizationchamber.

EXAMPLE 19 Effect of NO Gas Pressure on Sterilization Efficacy

A low pressure gauge is fitted to the sterilization chamber tubing. Athree-way stopcock (lure lock) is adapted to the gauge directly or via ashort length of tubing. From there a vacuum can be pulled with a 60 ccsyringe or pump if required. The chamber can be sealed with thestopcock, thus maintaining the vacuum. The NO gas pressure used for thesterilization trial is regulated by altering the mass ofdiazeniumdiolate in the gas generation chamber from the normal level of6.8 gm per 1000 cc of volume. Sterilizations are attempted using 1.7,3.4, 6.8 gms (control) of diazeniumdiolate in the gas generationchamber, keeping the 10:1 ratio of oxalic acid throughout theexperiment. Dead space is also accounted for. A successful outcome is 0CFU/ml in three trials. One failure (positive B. subtillis var nigerculture) in three trials is considered a failure at that measurement,therefore setting the limit at the measured parameter.

EXAMPLE 20 The Effect of Ambient Air on Sterilization Efficacy

The inclusion or exclusion of ambient air is a critical parameter, asthe ultimate mechanism of NO sterilization in this method can includethe formation of nitrous acid (HNO₂) on surface condensates. A smallpercentage of ambient air may be advantageously included in the process.The small amount of O₂ dissolved in a humid condensate can also sufficeto produce enough nitrous acid under conditions used in the method ofthe present invention.

A low pressure gauge is fitted to the sterilization chamber tubing. Athree-way stopcock (lure lock) is adapted to the gauge directly or via ashort length of tubing. From there a vacuum can be pulled with a 60 ccsyringe or pump if required. The chamber can be sealed with thestopcock, thus maintaining the vacuum. Graduated syringes filled withambient air can be attached to the stopcock and a known quantity of aircan be added to the sterilization chamber. The amount ofdiazeniumdiolate in the gas generation chamber is adjusted based on 2mol of NO per mol of diazeniumdiolate and using the Ideal Gas Law toreach what would be 1 ATM in 1 L, or 6.8 gm per liter of volume. Deadspace is determined and be accounted for with diazeniumdiolate mass.Volumes of ambient air representing 1, 2.5, 5, 10, 15, and 20% in thesterilization chamber is tested. These trials are performed at 25° C.,and 2 relevant extremes of temperature determined from experimentsoutlined above. Zero growth from B. subtilis contaminated items from theMaterial Panel in three trials is considered a successful outcome. Onefailure (positive B. subtillis var niger culture) in three trials isconsidered a failure at that measurement therefore setting the limit atthe measured parameter. A failure at the low level(s) of ambient air maybe an indication that oxygen is required, thus providing furtherevidence that the mechanism of action for NO in this process may berelated to the formation of nitrous acid.

The potential for an interdependency between ambient air and humidityhas been previously discussed.

EXAMPLE 21 Sterilization using a Variety of Oxides of Nitrogen

A mixture of nitric oxide and air will react, resulting in a mixturecontaining many different oxides of nitrogen. The concentration of eachnitrogen-oxide species that is present in a mixture will vary withtemperature, pressure, and initial concentration of the nitric oxide.The concentrations of various nitrogen-oxide species in air can bemeasured directly or predicted using established methods.

For example, the detailed chemical kinetics of NO oxidation in humid aircan be simulated using available chemical kinetics software (such asCHEMKIN software) and kinetics data found in the literature. In oneapproach to predict the composition and concentrations of species thatresult from a NO-air mixture, a closed homogeneous batch-reactor modelis used.

The results of the analysis for two different initial concentrations ofNO are shown in Table 6 below. These results show that after fiveminutes, the NO is oxidized to NO2, N2O4, nitric acid, nitrous acidHNO3, and smaller amounts of N2O3 and N2O5.

TABLE 8 Predicted Concentrations of Oxides of Nitrogen at RoomTemperature (75° F.) and 70% Relative Humidity Initial Mole Fraction NOin Air 3.00E−02 10.00E−02 Mole Fraction NO (5 minutes) 7.51E−04 1.11E−03 Mole Fraction NO2 (5 minutes) 2.24E−02  5.87E−02 Mole FractionN2O4 (5 minutes) 3.68E−03  2.39E−02 Mole Fraction HONO2 (5 minutes)3.50E−05  1.18E−04 Mole Fraction HNO2 (5 minutes) 2.91E−08  5.49E−08Mole Fraction N2O3 (5 minutes) 3.87E−06  1.41E−05 Mole Fraction N2O5 (5minutes) 2.01E−11  2.16E−10 Mole Fraction H2O (5 minutes) 2.01E−02 1.96E−02

Another embodiment of the system and method of the present inventionuses a sterilant gas comprised of nitrogen dioxide and/or other oxidesof nitrogen such as di-nitrogen tetroxide. These oxides of nitrogen canbe generated from nitric oxide and air supplied by the gas generatingchamber. In another embodiment, nitrogen dioxide and/or di-nitrogentetroxide can be delivered from a pressurized tank. NO₂ or N₂O₄ can bestored in high concentrations in a tank or can be diluted to lowerconcentrations in a mixture with either an inert gas, such as nitrogenor argon. Alternatively, the NO₂ or N₂O₄ could be diluted with air.

The gas or gas mixture can be delivered to the sterilization chamberthrough a metering regulator in fluid connectivity with thesterilization chamber or other gas delivery method known to one skilledin the art.

EXAMPLE 22 Sterilization Effectiveness Using a Variety of Oxides ofNitrogen

The experiments are conducted in glass vessels in which gases can bemetered in exact amounts. The tests were conducted using biologicalindicators made of stainless steel disks inoculated with 10⁶ bacillusstearothemophilus spores. The BI disks are heat-sealed in individualTYVEK sterilization pouches and placed in a glass vessel. The vessel isfilled with various mixtures of gases following specific protocols forthe order and timing of gas introduction.

In one protocol, the glass vessel is evacuated to 3″ Hg absolute. EitherNO or NO₂, as the sterilant gas, is added in an amount that correspondsto a 5% concentration (by volume) when the vessel is completely filledwith sterilant gas and diluent gas. After 5 minutes, either air ornitrogen was added to the vessel until atmospheric pressure is reached.The BI is exposed to the air or nitrogen for 10 minutes. Thereafter, theBIs, in their pouches, are removed from the vessel and taken to amicrobiological hood. The biological indicators are removed from thepouches and placed in tubes with sterile tryptic soy broth, incubated atfrom about 55 to about 60° C., and monitored for growth.

The results of a spore study is shown in Table 9.

TABLE 9 Results of Spore Studies in NO and NO₂ 5% NO₂ in 5% NO in 5% NO₂in Air with Air with N₂ with Exp Humidity Humidity Humidity Time ChipChip Chip 10 min Dead Dead Dead Control Live Live Live

EXAMPLE 2 Sterilization Cycles Including Exposure Periods withParticular Oxides of Nitrogen

The ability of the nitrogen oxide gases to penetrate an inner open spaceor cavity within a tube (i.e., a lumen) and inactivate spores in thelumen is evaluated. The spores used are those that are most resistant tothe nitrogen oxide gases.

The following configuration of lumens are tested:

(a) 17″ stainless (SS) tubes having a 2.5 mm inner diameter (I.D.), bentin “U” shape

(b) 60″ polyethylene (PE) tube having a 4.5 mm ID, coiled and in Tyvekpouch

(c) 60″ PE tube having a 4.5mm ID and a 1.5 mm outer diameter (O.D.)PEEK tube inside, coiled and in Tyvek pouch

Wire biological indicators are inoculated with 10⁶ Bacillusstearothemophilus spores (ATCC 7953), (Raven Lot 3W67583) and placed atthe center point of the tube in each of these configurations. The coiledpolyethylene tubes were then heat-sealed in a TYVEK sterilization pouch.

Separately, each of the lumen samples are tested. The stainless steeltube (triplicate samples: SS-1, SS-2, and SS-3), the polyethylene tube,and the polyethylene tube with the PEEK catheter were each placed in aresistometer and exposed to the following gas cycle for 5 minutes at 23°C. This gas cycle is defined as follows:

The sterilization chamber/resistometer is evacuated and held at vacuumfor 2 minutes. NO gas is then added to the sterilization chamber untilthe vacuum is decreased by 1″Hg. This condition is maintained for 2minutes. Are is then added until the chamber reaches atmosphericpressure and the condition is held for 5 minutes.

The results of the spore studies are shown in Table 10. After 14 days ofincubation, bacterial growth is seen in all of the control samples andin none of the exposed samples after 5 minutes of exposure to the gas.

TABLE 10 Results of Spore Studies in Lumens (c) (b) PE Sheath Exposure(a) (a) (a) PE with Time SS-1 SS-2 SS-3 Sheath Catheter 5 min Dead DeadDead Dead Dead 5 min Dead Dead Dead Dead Dead 5 min Dead Dead Dead DeadDead Control Live Live Live Live Live

EXAMPLE 24 Effect of Insoluble Crystal Occlusion of SterilizationEfficacy

Numerous studies have demonstrated the difficulty, especially withgaseous sterilizing agents, of killing spores occluded within waterinsoluble crystals. See, for example, Abbott C F et al., J PharmPharmacol 8:709-720, 1956; Doyle J E and Ernst R R, Applied Microbiology15(4): 726-730, 1967. The method of Doyle and Ernst is used for theproduction and isolation of spores, occlusion of spores in crystallinecalcium carbonate, and recovery of occluded spores for determination ofsterilization efficacy.

Ten ml solution of 1.11% CaCl₂ is prepared containing 10⁶ spores per ml.To this, ten ml of 1.06% Na₂CO₃ is added rapidly and the mixture isshaken vigorously. Crystals of Ca₂CO₃ will form immediately, occludinglarge quantities of spores per crystal. The crystals are washed usingdistilled water in three 20,000×g centrifugation steps. The crystals arebrought back to 10 ml in distilled water and 0.2% methylcellulose foreasy handing. Ten μl of the crystal suspension is blotted onto strips offilter paper, dried at room temperature, and further dried at 90° C. for16 hrs.

After exposure to the sterilization agent, the paper strips are placedin 25 ml of sterile 3.0% NH₄Cl for 3 days at 0° C. to dissolve thecrystals. The strip and solution is placed in a blender, followed bysonication for 5 minutes. The samples are diluted and plated on tryptoneglucose yeast extract agar for counting. Zero growth in three separateexperiments is considered a successful outcome.

EXAMPLE 25 Sterilization Efficacy in Devices Having Long, Narrow andDead-End Lumens

Many studies have documented the difficulties in reliably sterilizinglong, narrow, and dead-end lumens. See, for example, Alfa M C., InfectControl Ster Technol April: 26-36, 1997; and Rutala W A et al., InfectControl Hosp Epidemiol 19: 798-804, 1998. To test the ability of thissterilization process to effectively sterilize these types of devices,non-porous Teflon tubing (≦3 mm LD.) are cut into 125 cm lengths and aculture of B. subtillis var niger (10⁶ CFU/ml) is pushed through thetubing using a 60 ml syringe. The tubing is drained and allowed to airdry. Some tubing is plugged at one end with a tight fitting plug. Thegas tightness of the plug is tested by applying a small amount of airpressure using a 60 cc syringe. Alternate methods of sealing one end ofthe tubing include heat sealing, solvent welding, and clamping. Open orsealed end tubing is coiled with care to not crimp the tubing and placedin the sterilization chamber for processing. After the sterilizationprocess is complete, the tubing is cut into 4 inch sections and placedinto sterile culture tubes containing enough LB to completely submergethe tubing sections. Sterilization efficacy is evaluated as describedabove.

EXAMPLE 26 Sterilization Efficacy in Instruments with Occluded Joints

Surgical scissors and forceps are contaminated to beyond the swiveljoint by dipping in contaminated broth at 10⁶ CFU/ml. The swivel jointis actuated while the instrument is submerged in bacterial broth toallow bacteria to get between the arms of the instrument. The instrumentis allowed to air dry, and subject to the sterilization process. Zerogrowth in three separate trials is considered a successful outcome.

EXAMPLE 27 Sterilization of Instruments in Individual SterilizationPouches

Surgical scissors and forceps are contaminated to beyond the swiveljoint by dipping in contaminated broth at 10⁶ CFU/ml. The instrumentsare dried and sealed in a V. Mueller™ Dual Peel Seal Pouch FisherScientific) and inserted into the sterilization chamber of the devicefor processing. After processing, the contaminated forceps are carefullyremoved from the pouches using sterile technique and under sterileconditions, and placed in a culture flask containing sterile LB media,and sterilization efficacy is assessed as described above. Zero growthin three separate trials is considered a successful outcome. Otheritems, such as long narrow lumen tubing may also be added to thisprotocol for study.

EXAMPLE 28 Two Stage Operating Cycle

The overall sterilization effectiveness is dependant on the ability ofthe gas to come in contact with the microbes, and the effectiveness withwhich the sterilizing gases kill the microbes they contact. In onepreferred embodiment of the sterilizing method of the present invention,the operating cycle for the sterilizing sterilizer maximizes both thepenetration of sterilizing gases into devices, as well as the killing ofthe microorganisms that are contacted. The penetration of a gas into thesmall crevices, lumens, gaps, cracks, mated surfaces, and interiorsurfaces of medical devices is dependant (among other things) on the gasmolecule size, diffusivity, “stickiness”, and tendency to be absorbedinto or adsorbed onto the surface of solid or liquid materials.

It has been found that NO has better transport properties than NO₂because it is a smaller, less “sticky” molecule. In a preferredembodiment of the present invention, the sterilization cycle involves atwo stage approach. During the first stage, the NO is allowed topenetrate throughout the surfaces of the object to be sterilized. Duringthe second stage, the NO is oxidized to form NO₂ and other oxides ofnitrogen. The NO₂ and other oxides of nitrogen offer additional,effective modes of microbial kill. This two stage approach minimizes theamount of sterilizing gas volume and the time that is needed tosterilize a device that has hidden surfaces and/or a complex geometry.

One example of this preferred two stage operating cycle is conducted asfollows. The device to be sterilized is placed in a chamber capable ofgenerating and maintaining a vacuum as well as receiving sterilizinggases and air. The sterilization chamber is sealed. The sterilizationchamber is evacuated to a vacuum level of less than 3″Hg absolute. NOgas is introduced to the evacuated chamber in an amount that correspondsto 1-8% concentration in the final sterilizing gas mixture. Theconditions are maintained for a period of time from about 30 seconds toabout 5 minutes. Humidified air is added until the chamber reachesatmospheric pressure. The conditions are maintained for a period of timefrom about 30 seconds to about a few hours, depending on the sterilantconcentration. The sterilization chamber is evacuated and purged withair before the sterilized device is removed.

This two stage operating cycle is more effective at sterilizing objecthaving a lumen than both (1) a cycle that introduces NO and airsimultaneously and (2) a cycle that introduces NO₂ in air or nitrogen.

EXAMPLE 29 Sterilization of Polymers

To determine if a polymer, the polymer is inoculated with the sporesolution, dried and exposed to the oxides of nitrogen. After exposure,the spores are washed off the polymer into a growth medium and incubatedto assess growth.

The Effect of Nitric Oxide Sterilization Methods on BioresorbablePolymers.

This Example evaluates the molecular weight profile of polyester-basedbioresorbable polymers before and after sterilization treatment overtime. Specifically, the effect of three sterilization techniques areevaluated: ethylene oxide (EtO) treatment, gamma irradiation and themethod of the present invention using oxides of nitrogen. The polyestermaterials include LACTEL® DLPLG (poly-DL-lactide-co-glycolide, 50/50),DLPLA (poly-DL-lactide-COOH), LPLA (poly-L-lactide) and PCL(poly-e-caprolactone) polymers purchased from the DURECT Corporation(Pelham, Ala.). Treated samples are evaluated immediately aftertreatment (Day 0). Samples are treated using the method of the presentinvention (or industry standard conditions for EtO (100% EtO for 1 hour,57° C., ≧70% RH, followed by 15 hour aeration) and gamma irradiation(26.8 kGy). For each sterilization method, untreated control samples ofeach polymer are prepared.

For all samples treated with NO_(x), there is no significant change inthe MW profile of the bulk polymer. In a few samples there are somesmall differences noted at the lower MW region (about 10 min retentiontime) of the chromatogram. The EtO treated samples change shape relativeto the control samples, are difficult to remove from the Tyvek®sterilization pouch, and adhere to the surface of the bag. There is nosignificant change in the MW profile of the bulk polymer. For allirradiated samples tested there is a detectable change in the MW profileof the bulk polymer.

Three sterilization treatments (NO_(x), EtO and irradiation) are appliedto bulk samples of four polyester bioresorbable polymers: LACTEL® DLPLG(poly-DL-lactide-co-glycolide, 50/50), DLPLA (poly-DL-lactide-COOH),LPLA (poly-L-lactide) and PCL (poly-e-caprolactone). The polymer samplesare tested upon receipt (identified as Day 0, time point 1 by GPCChromatography to analyze their molecular weight. Samples treated withthe oxides of nitrogen exhibit no significant difference in their MWprofile, as compared to the control samples. Although the EtO treatedpolymer samples display no significant difference in their MW profile,they become visually deformed relative to the control samples and stickto the sterilization pouch. The gamma irradiated polymer samples displaya detectable change in the MW with the average molecular weight shiftingto a lower value which suggests fragmentation of the bulk polymerchains. The gamma irradiated change is most pronounced for the PCLsamples.

EXAMPLE 30 Protein Sterilization

Under sterilization conditions known to kill stearothermophilus spores,a sample protein is tested to determine if the sterilization conditionsaffect the protein's biological function. Trypsin is used as the sampleprotein. The trypsin is in powder form during the sterilization cycleand thereafter reconstituted in solution and tested for itsfunctionality. A biological indicator is included in the samesterilization container as the protein powder.

In describing representative embodiments of the invention, thespecification may have presented the method and/or process of theinvention as a particular sequence of steps. However, to the extent thatthe method or process does not rely on the particular order of steps setforth herein, the method or process should not be limited to theparticular sequence of steps described. As one of ordinary skill in theart would appreciate, other sequences of steps may be possible.Therefore, the particular order of the steps set forth in thespecification should not be construed as limitations on the claims. Inaddition, the claims directed to the method and/or process of theinvention should not be limited to the performance of their steps in theorder written, and one skilled in the art can readily appreciate thatthe sequences may be varied and still remain within the spirit and scopeof the invention.

The foregoing disclosure of the embodiments of the invention has beenpresented for purposes of illustration and description. It is notintended to be exhaustive or to limit the invention to the precise formsdisclosed. Many variations and modifications of the embodimentsdescribed herein will be apparent to one of ordinary skill in the art inlight of the above disclosure.

1. A method for sterilizing an object in a gas-tight sterilizing chamberusing a sterilant gas consisting essentially of NO₂ comprising: placingthe object in the gas-tight sterilizing chamber; providing a selectedamount of the sterilant gas to the sterilizing chamber; after supplyingthe sterilizing chamber with the selected amount of the sterilant gas,providing a humidified gas into the sterilizing chamber to form adiluted, humidified sterilant gas in the sterilizing chamber; andperforming a dwelling operation at about room temperature for a selectedperiod of time during which the diluted, humidified sterilant gassterilizes the object.
 2. The method of claim 1, wherein the humidity ofthe diluted, humidified sterilant gas in the sterilant chamber is fromabout 40% to about 80%.